spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online August 25, 2006
doi: 10.1242/10.1242/dev.02535

Development 133, 3575-3585 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Imai, A.
Right arrow Articles by Takahashi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Imai, A.
Right arrow Articles by Takahashi, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene

Akihiro Imai1,2, Yoshie Hanzawa2,*, Mio Komura1,2, Kotaro T. Yamamoto2, Yoshibumi Komeda2,{dagger} and Taku Takahashi1,{ddagger}

1 Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, N10, W8, Sapporo 060-0810, Japan.


Figure 1
View larger version (19K):

[in a new window]
  		Fig. 1. Height comparisons between wild-type (WT), acl5, acl5 heterozygous for sac (sac/+), acl5 homozygous for sac, and sac plants. The heights were measured in 6-week-old plants. Bars show mean±s.d. (n=10).

 

Figure 2
View larger version (57K):

[in a new window]
  		Fig. 2. Morphological phenotypes of sac acl5 mutants. (A) From left to right, 6-week-old wild-type (WT), acl5 and sac acl5 plants. (B,C) Longitudinal (B) and transverse (C) sections of the first internode of wild-type (WT), acl5 and sac acl5 inflorescence stems. Scale bars: 200 µm. (D) Quantitative RT-PCR analysis of the HD-Zip III class genes. Total RNA was prepared from whole seedlings of 12-day-old wild-type (WT), acl5 and sac acl5 plants. Levels of the ACTIN8 (ACT8) transcript were used as a reference; values are expressed as ratios to the transcript level of each gene in the wild-type seedlings. Bars show mean±s.d. (n=3).

 

Figure 3
View larger version (81K):

[in a new window]
  		Fig. 3. Northern analysis of EXGT-A1, {gamma}-TIP,ACL5 and SPMS expression levels. Total RNA was prepared from apical meristems and flower buds of 6-week-old wild-type (WT), acl5, and sac acl5 plants. Each lane contains 10 µg of total RNA. rRNA is shown as a loading control.

 

Figure 4
View larger version (24K):

[in a new window]
  		Fig. 4. Genetic interactions of sac with phytohormone-related and spms mutants. (A) Effect of each sac allele on the plant heights of 8-week-old axr2, gai and dim mutants. (B) Effect of each sac allele on the plant heights of 6-week-old acl5 and acl5 spms mutants. Bars show mean±s.d. (n=6).

 

Figure 5
View larger version (62K):

[in a new window]
  		Fig. 5. Map-based cloning and sequence analysis of SAC51. (A) The region of chromosome 5 containing SAC51. The chromosome is depicted by the uppermost horizontal line with the franking markers LFY3 and m555. Below this are three P1 or BAC clones: MHJ24, MSJ1 and T12B11. The markers (see Materials and methods) and number of recombinants are shown. (B) Structure of the SAC51 gene. Boxes indicate exons and the solid lines between boxes indicate introns. A black box represents a principal ORF and gray boxes represent uORFs. The location of the sac51-d mutation is shown. (C) The wild-type (Col-0) genomic DNA sequence of SAC51 in which the regions corresponding to the cDNA shown in uppercase letters (GenBank Accession number: AY062561). The deduced amino acid sequences of the uORFs and main ORF are indicated below the nucleotide sequences. Asterisks indicate stop codons and the arrowhead indicates the position of the sac51-d mutation. The SAC51 bHLH domain is boxed. (D) Alignment of the bHLH domain of SAC51, its homolog At5g09460 and three characterized proteins from Arabidopsis: SPATULA (SPT) (Heisler et al., 2001Go), PHYTOCHROME INTERACTING FACTOR3 (PIF3) (Ni et al., 1998Go) and TRANSPARENT TESTA8 (TT8) (Nesi et al., 2000Go). Black blocks indicate residues identical to the SAC51 sequence; gray blocks indicate similar amino acids.

 

Figure 6
View larger version (75K):

[in a new window]
  		Fig. 6. Northern analysis of SAC51 expression levels. (A) Tissue profiling of SAC51 transcripts. Total RNA was prepared from 12-day-old seedlings (Sd), and from 6-week-old plant leaves (Lf), stems (St), roots (Rt), flowers (Fl) and green siliques (Sq). (B) SAC51 transcript levels in wild-type (WT), acl5, and sac acl5 plants. Total RNA was prepared from 12-day-old seedlings. Each lane contains 10 µg of total RNA and rRNA is shown as a loading control.

 

Figure 7
View larger version (30K):

[in a new window]
  		Fig. 7. Effects of the SAC51 5'-leader on GUS reporter gene expression. (A) The GUS gene constructs consisting of the SAC51 promoter with either wild-type SAC51 or the mutant sac51-d 5'-leader sequences and the GUS coding sequence. White arrows represent the SAC51 promoter, boxes indicate exons, lines indicate introns; gray boxes represent uORFs and black blocks correspond to the GUS-coding sequence. (B-F) GUS staining (blue) in transgenic plants harboring the SAC51-GUS construct (left) or the sac51-d-GUS construct (right). Sixteen-day-old seedlings (B), inflorescences (C), young rosette leaves (D), roots (E), and mature embryos (F) are shown. Scale bars: B,C, 5 mm; D,E, 1 mm; F, 100 µm. (G) The relative GUS translational efficiency of SAC51-GUS and sac51-d-GUS constructs in wild-type (WT), acl5-1 and sac51-d acl5-1 backgrounds, and that of the sac51-C549A-GUS (C549A) construct in the wild-type background. The levels of GUS activity and GUS mRNA in the SAC51-GUS transgenic line in the wild-type background were set at 1.0. The GUS translational efficiencies were calculated as the GUS activity divided by the GUS mRNA level for each plant. The GUS mRNA levels were normalized to the ACT8 level in each sample. Bars show mean±s.d. (n=3). (H) Nucleotide and amino acid changes at the sac51-d mutation site in SAC51-GUS, sac51-d-GUS and sac51-C549A-GUS constructs. Nucleotide and amino acid changes are in bold.

 

Figure 8
View larger version (26K):

[in a new window]
  		Fig. 8. A model of uORF-mediated translational control of SAC51 expression via ACL5 function. SAC51 and sac51-d transcripts are represented as horizontal lines, the fourth uORF as a dark gray box, uORFs (except the fourth uORF) as light gray boxes, the main ORF as a white box and SAC51 bHLH proteins as hatched circles. Small (40S) and large (60S) ribosomal subunits are indicated by small and large black circles, respectively.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006